Composition and use of macro-minerals to lower postprandial glycemic response and reduce body weight

ABSTRACT

Composition and use of macro-minerals to lower postprandial glycemic response and reduce body weight are disclosed herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional Application Ser.No. 62/308,546, filed Mar. 15, 2016, which is hereby incorporated byreference in its entirety.

BACKGROUND

The invention generally relates to food products, more specificallybread products.

Postprandial glycaemia or glycemic index (GI) is known to be positivelyassociated with the development of diabetes and obesity. The latter isalso the result of imbalance between energy intake and expenditure.Furthermore, intake of refined carbohydrates (e.g. made from white wheatflour, white rice and byproducts, sugar, sweeteners, etc.) is positivelyassociated with the development of obesity and diabetes. Theavailability of refined carbohydrates increased tremendously during thepast few decades due to modernization and its related factors; includingindustrialization and globalization of food markets, and refinedcarbohydrates seem to contribute to more than 50% of the food supply(kcal per capita per day) in most countries. Thus, there is a paramountneed to uncover the detrimental potential of refined carbohydrates.Refinement of grains removes a large percentage of their vitamin andmineral contents (around 70%). However, many countries tend to enrichtheir white flour with several vitamins, known to be involved incarbohydrate metabolism (e.g. thiamin, riboflavin and niacin). Whileenrichment with minerals; especially macro-minerals, has been ignoreddespite their ultimate importance.

On the other hand, most studies on obesity were mainly focused on theintake and metabolism of macronutrients (carbohydrates, proteins andfats), though their absorbed forms (type of fatty acids, amino acids,glucose, etc.) are not believed to have been drastically altered, incontrast to that of micronutrients.

The present invention solves these problems as well as others.

SUMMARY OF THE INVENTION

Provided herein are compositions and use of macro-minerals to lowerpostprandial glycemic response and reduce body weight. A formulation offood products supplemented with macro-minerals is disclosed andgenerally comprises: a food product including a carbohydrate that lowersthe glycemic response including Phosphorus (P) at an activeconcentration.

A method of preventing weight gain and reducing waist circumference isdisclosed and generally comprises: delivering at least between about 300mg to about 500 mg of phosphorus with each meal, wherein each mealinclude about 300 to about 500 Kcal of carbohydrate over a period oftime and increasing energy expenditure.

A method of enriching a bread product is generally disclosed andcomprises: restoring the levels of phosphorus (P), potassium (K), andmagnesium (Mg) prior to processing and milling, whereby each Kg of whiteflour contained between about 2.0 and 5.0 g of Mg and between about 5.0and about 10.0 g of K and P; and fortifying the bread product to doublethe premilling levels of phosphorus (P), potassium (K), and magnesium(Mg).

The methods, systems, and apparatuses are set forth in part in thedescription which follows, and in part will be obvious from thedescription, or can be learned by practice of the methods, apparatuses,and systems. The advantages of the methods, apparatuses, and systemswill be realized and attained by means of the elements and combinationsparticularly pointed out in the appended claims. It is to be understoodthat both the foregoing general description and the following detaileddescription are exemplary and explanatory only and are not restrictiveof the methods, apparatuses, and systems, as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

In the accompanying figures, like elements are identified by likereference numerals among the several preferred embodiments of thepresent invention.

FIGS. 1A-1C are graphs showing the Changes in Serum Phosphorus (a),Glucose (b) and Insulin (c) levels of subjects in experiment 1;#Experiment 1: After the ingestion of 500 mg phosphorus (-♦-), 75 gglucose (-▴-) or co-ingestion glucose+phosphorus (75 g glucose+500 mg ofphosphorus) (..□..), where * p-value <0.05: Paired t-test in the sametreatment in comparison with baseline (time 0) value, where FIG. 1A hasa p-value <0.05: Paired t-Test, phosphorus vs glucose treatments at eachtime point, where FIG. 1B the p-value <0.05: Paired t-Test, phosphorusvs glucose+phosphorus treatments at each time point, where FIG. 1C thep-value <0.05: Paired t-Test, glucose vs glucose+phosphorus treatmentsat each time point.

FIGS. 2A-2C are graphs showing the Changes in Serum Phosphorus (FIG.2A), Glucose (FIG. 2B), and Insulin (FIG. 2C) levels of subjects inexperiment 2. #Experiment 2: After the he ingestion of 75 g glucose 60min after placebo (-♦-) or 500 mg phosphors (..□..) preload; * p-value<0.05: Paired t-test in the same treatment in comparison with baseline(time −60 min) value. A p-value <0.05: Paired t-Test between placebo andphosphorus preload treatments at each time point.

FIGS. 3A-3C are graphs shows the postprandial energy expenditure of mealcontaining P was significantly higher than that of placebo (p=0.007),this increase was associated with a significant rise in fat oxidation(%) (p=0.022, FIG. 3C), while carbohydrate oxidation (%) was decreased(p=0.023, FIG. 3B).

FIG. 4 is a Study flow diagram.

FIG. 5 is a Table showing the baseline characteristics of the studyparticipants.

FIG. 6 is a Table showing the changes in anthropometric and biochemicalcharacteristics from baseline to 12 weeks.

FIG. 7 is a Table showing the changes in subjective appetite scores frombaseline to 12 weeks.

FIGS. 8A-8C are graphs showing the Postprandial changes (Δ) in thelevels of total serum phosphorus (FIG. 8A), magnesium (FIG. 8B) andpotassium (FIG. 8C) following the ingestion of the different types ofpita breads [White pita bread: WP (- -▴- -); White pita bread-restored:WP-R (--▪--); White pita bread fortified: WP-F (-♦-). * Values aresignificantly different from control test food (WP) within a specifictime at p<0.05, as analyzed by paired t-test.

FIGS. 9A-9B are graphs showing the Postprandial changes (Δ) in thelevels of serum triglycerides (FIG. 9A) and glucose (FIG. 9B) followingthe ingestion of the different types of pita breads. [White pita bread:WP (- -▴- -) White pita bread-restored: WP-R (--▪--); White pita breadfortified: WP-F (-♦-)]; *, **, *** values are significantly differentfrom control test food (WP) within a specific time at p<0.05, p<0.01,and p<0.001 respectively, as analyzed by paired t-test.

FIG. 10 is a graph showing the Glycemic index of supplemented pitabreads as compared to white pita bread. WP: white pita bread; WP-R:white pita bread-restored; WP-F: white pita bread fortified. * Valuesare significantly different from white pita bread at p<0.05, as analyzedby paired t-test.

DETAILED DESCRIPTION OF THE INVENTION

The foregoing and other features and advantages of the invention areapparent from the following detailed description of exemplaryembodiments, read in conjunction with the accompanying drawings. Thedetailed description and drawings are merely illustrative of theinvention rather than limiting, the scope of the invention being definedby the appended claims and equivalents thereof.

Embodiments of the invention will now be described with reference to theFigures, wherein like numerals reflect like elements throughout. Theterminology used in the description presented herein is not intended tobe interpreted in any limited or restrictive way, simply because it isbeing utilized in conjunction with detailed description of certainspecific embodiments of the invention. Furthermore, embodiments of theinvention may include several novel features, no single one of which issolely responsible for its desirable attributes or which is essential topracticing the invention described herein. The words proximal and distalare applied herein to denote specific ends of components of theinstrument described herein. A proximal end refers to the end of aninstrument nearer to an operator of the instrument when the instrumentis being used. A distal end refers to the end of a component furtherfrom the operator and extending towards the surgical area of a patientand/or the implant.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention are to be construed to cover boththe singular and the plural, unless otherwise indicated herein orclearly contradicted by context. It will be further understood that theterms “comprises,” “comprising,” “includes,” and/or “including,” whenused herein, specify the presence of stated features, integers, steps,operations, elements, and/or components, but do not preclude thepresence or addition of one or more other features, integers, steps,operations, elements, components, and/or groups thereof.

Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein. The word “about,” when accompanying anumerical value, is to be construed as indicating a deviation of up toand inclusive of 10% from the stated numerical value. The use of any andall examples, or exemplary language (“e.g.” or “such as”) providedherein, is intended merely to better illuminate the invention and doesnot pose a limitation on the scope of the invention unless otherwiseclaimed. No language in the specification should be construed asindicating any non-claimed element as essential to the practice of theinvention.

References to “one embodiment,” “an embodiment,” “example embodiment,”“various embodiments,” etc., may indicate that the embodiment(s) of theinvention so described may include a particular feature, structure, orcharacteristic, but not every embodiment necessarily includes theparticular feature, structure, or characteristic. Further, repeated useof the phrase “in one embodiment,” or “in an exemplary embodiment,” donot necessarily refer to the same embodiment, although they may.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and nutraceutical, and food arts.

Phosphorus is essential for the metabolism of carbohydrates (glucose),especially for the intracellular trapping of glucose. The entrapment ofglucose thus induces several desired effects including: 1) Glucose isout of the blood circulation, 2) Energy or ATP can then be released, 3)With the attainment of energy, the body no more feels the need for foodintake and thus experiences a state of suppressed appetite.

In humans, the availability of free phosphorus is limited and thiscreates a competition between different metabolic processes that requirephosphorus. Such competition is believed to compromise the activities ofseveral metabolic processes especially in the postprandial status whenthe need is heightened. The present invention comprises carbohydrateingestion (glucose) accompanied by exogenous availability of phosphorusin order to fulfill the requirement of all metabolic processes and suchavailability would improve postprandial glycaemia and decrease bodyweight. A reduced glycemic index is a glycemic index value that is atleast 5% lower after ingestion of an equivalently sweet amount ofsweetener in a product as described herein relative to the index of agiven amount of sucrose when ingested as refined sucrose. The reductionin glycemic index is preferably greater, e.g., about 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90% or more.

In one embodiment, phosphorus compounds are added to food products as toincrease the phosphorus percentage by at least about 50% to about 500%,or potassium compounds are added to food products to increase thepotassium percentage by at least 50% to about 500%, or magnesiumcompounds are added to food products to increase the magnesiumpercentage by at least 50% to about 500%. Alternatively, potassiumcompounds can be delivered in form of supplements to be added to foodproducts in order to exert its beneficial effect. Supplements may takethe form of tablets, pills, sachets, capsules, powders, liquid forms.

Phosphorus alone or with magnesium and potassium can be used to enrich(fortify) refined carbohydrates, food products, bread products, andtheir byproducts (wheat flour and products, sugar sweeteners, corn flourand products, white rice and products). Phosphorus compounds may beadded in the amounts of between about 500 mg per 500 kcal carbohydratemeal.

The present invention comprises the addition of phosphors to an oralglucose load to improve postprandial insulin sensitivity (decreasing thepostprandial serum glucose concentration and postprandial insulinconcentration, Example 1). The addition of phosphorus may be in the formof potassium phosphate in the amount between about 100 mg and about 500mg in a tablet or pill form. In one embodiment the present inventioncomprises ingesting phosphorus compounds at least between about 30minutes to about 90 minutes before eating or ingesting any glucose. Thedecrease of postprandial serum glucose concentration may be betweenabout 2 and 10 mg/dl. The decrease in serum glucose levels occurs duringa time period. The decrease in serum insulin may between about 10 μU/mland 40 μU/ml. The increase in insulin sensitivity may between about 7.00and 20.00 according to the insulin sensitive index.

The present invention comprises the addition of phosphorus tocarbohydrate preloads was able to decrease subsequent energy intake(Example 2). The present invention comprises the addition of betweenabout 100 mg and 600 mg of phosphorus to a solution and taken before ameal. In one embodiment, the phosphorus is a mixture of potassium andsodium phosphate. In one embodiment, the time before the meal is betweenabout 30 and 90 minutes. The addition of phosphorus to the water preloadmay comprise at least between about a 10% to a 40% reduction in energyintake at the subsequent meal, and at least between about a 10% to 40%reduction in glucose, while the addition of phosphorus to the sucrose orfructose preloads comprises at least between about a 10% to 40%reduction in energy intake at the subsequent meal.

The present invention comprises the addition of phosphorus to a highcarbohydrate meal was able to increase and prolong postprandialthermogenesis (energy expenditure) which was, interestingly, caused byan increase in fat oxidation (Example 3).

The present invention comprises the ingestion of phosphorus tablets withthe main meals (breakfast, lunch and dinner) for at least 12 weeks toreduce weight and waist circumference, and to decrease appetite (Example4). The present invention comprises the ingestion of at least betweenabout 300 mg to about 500 mg of phosphorus with each meal, wherein eachmeal include about 300 to about 500 Kcal of carbohydrate (or about 1 mgof phosphorus per 1 Kcal from refined carbohydrate) over a period oftime prevents weight gain and reduce waist circumference amongoverweight and obese adults. The period of time may be between about 10weeks to about 15 weeks. An overweight adult is an adult between theages of 18 to 45 with a Body Mass Index <25 kg/m². The decrease in bodyweight may be between about 0.40 kg to about 0.80 kg. The decrease inthe waist circumference is between about 2.00 cm to about 5.00 cm. Theserum levels of phosphorus during the time period will not be affected.The decrease in appetite may be between about −0.4 to about −1.5.

The present invention comprises the production of a bread product(Example 5) from flour enriched with phosphorus (P) plus two othermacronutrients potassium (K) and magnesium (Mg), which are depletedduring the process of flour milling at two levels. A first level is torestore the original levels of phosphorus (P), potassium (K), andmagnesium (Mg) (prior to processing and milling), whereby each Kg ofwhite flour contained between about 2.0 and 5.0 g MgCO₃ and betweenabout 5.0 and about 10.0 g of KH₂PO₄. Another level was fortified breadproduct to double the premilling levels of phosphorus (P), potassium(K), and magnesium (Mg). In one embodiment, the fortification compriseseach Kg of white flour containing between about 5.0 and about 10 g ofMgCO₃ and between about 20 and about 30 g of KH₂PO₄. In one embodiment,phosphorus compounds are added to bread products as to increase thephosphorus percentage by at least about 50% to about 500%, or potassiumcompounds are added to bread products to increase the potassiumpercentage by at least 50% to about 500%, or magnesium compounds areadded to bread products to increase the magnesium percentage by at least50% to about 500%. The present invention a) Reduced postprandialglycaemia between about 20% and 40% (GI) as compared to white pita breadwhereby Postprandial serum glucose change is lower between about 20% and40% than that of regular bread products at 60 minutes onwards; b)maintained low levels of postprandial plasma triglycerides between about−5 mg/dl and about −20 mg/dl; and c) palatability of pita bread was notaffected by the addition of macro-minerals. Maintained similar sensorycharacteristics using triangle difference and acceptability tests.

The present invention comprises phosphorus' co-ingestion with refinedcarbohydrates improves postprandial glycaemia, increase energyexpenditure, decrease appetite and decrease body weight. Moreover, itsaddition to bread did not affect the sensory properties andpalatability. The levels of phosphorus used, whether in the form oftablets or enrichment of bread, have marginal effects on the productioncosts of bread and its byproducts. Moreover, the daily ingestion ofabout 500 g (2000 Kcal) of the enriched bread, though the average USAdaily wheat flour consumption per capita is about 160 g, would be lowerthan the upper limit of intake (4000 mg per day) set by different healthagencies/organizations including institute of medicine (TOM).

The present invention comprises: 1) decreasing postprandial glycemiathrough improving insulin sensitivity), 2) increasing energy expenditureand 3) suppressing appetite. Consequently, the present inventionproduces better glycemic and weight control and enhances the health andwellbeing of populations.

The present invention comprises enrichment of refined carbohydrates orthe ingestion of phosphorus tablets or sachets with refined carbohydratemeals. The present invention comprises the enrichment of refinedcarbohydrates that includes a high level of adherence due to theirmarginal effects on palatability of foods and dietary practices/habitsof populations.

The present invention improves the health status of subjects by curbingthe detrimental effects of refined carbohydrates comprising: 1) Lowerspostprandial glycemic response of refined carbohydrates; 2) Increasesenergy expenditure following ingestion of refined carbohydrates; and 3)Decreases appetite; and 4) Reduces body weight of subjects.

The present invention comprises: 1) targeting of the general population;2) ensuring high compliance, when used as fortificant for refinedcarbohydrates given that they are highly consumed on regular basis (e.g.bread is a staple item in most peoples' diets); 3) delivering effectsthrough the use of an essential nutrient and within the normal range ofrequirement (dietary reference intake, i.e. a nutritional notpharmacological dose); 4) providing an approach for reducingpostprandial glycemia, appetite and body weight, as well as increasingpostprandial thermogenesis; 5) not affecting the characteristics ofbread and, therefore, would not require dietary changes; 6) minimallyaffecting the production costs of refined carbohydrates e.g. bread; 7)incorporating into food (e.g. bread) that has no effect on palatabilityand sensory properties.

The present invention decreases weight and waist circumference andcauses removal of fat rather than loss of lean body mass, especiallysince the increase in energy expenditure was mainly related to anincrease in fat oxidation. Therefore, the effect of phosphorus on bodycomposition and protein synthesis is shown. In addition, the impact ofphosphorus addition on several components of the metabolic syndromeespecially among subjects with an existing metabolic syndrome. Thepalatability and sensory properties of other refined carbohydrates andtheir byproducts (especially white rice and byproducts since it isstaple food in many countries) is not affected by the present invention.

The reduction of an excessive body fat causes effect to treat, improveand prevent a disease the cause of which is said to be corpulence orexcess of body weight, for example diabetes, arteriosclerosis,hyperpiesia, cancer, hyperlipemia, ryeumatism, hyperrucicemia, arthritisdeformans, gout, cerebral accident, ischemic heart disease, respiratoryinjury, pancreatitis, cataract, Alzheimer's disease, allergic disease,aging, hidrosis, ischemic disease, complications of diabetes beingkidney disease, nerve injury and retinopathy.

This invention has successfully identified the potential beneficial roleof minerals in improving the glycemic response of white bread andoverall glucose control in healthy male subjects. For the presentinvention, sensory results did not indicate any major effect of theexperimental treatments on the quality of the bread, which is anencouraging outcome and indicative of the tolerance of bread quality tothe addition of P, K and Mg.

Formulating a white bread supplemented with the proper levels ofmacro-minerals, as shown in this work/invention could be of huge valueto the glycemic response of this staple food and could serve as a modelfor other food products with high postprandial glycemic response,thereby lowering postprandial glycaemia of peoples' diets, an essentialstep and a major preventive measure for several non-communicablediseases.

Phosphorus may take the form of phosphorus-based acids, Monopotassiumphosphate, any phosphates (compounds containing the phosphate ion, PO₄⁻³), phospholipids, soluble salts of potassium, bisphosphonate, ahydroxybisphosphonate, a phosphonate, a phosphate, anaminomethylenephosphonic acid, and an acidic peptide. Polyphosphonicacids and aminomethylenephosphonic acids have a high affinity for bonein vivo due to their binding of the exposed calcium ions inhydroxyapatite (calcium phosphate), and also are suitable for use in thecontext of the present methods. The terms “phosphonate, phosphate, andaminomethylenephosphonate” are meant to encompass the phosphonic acids,the phosphoric acids, and aminomethylenephosphonic acids, respectively,as well as any salts, hydrolyzable esters, and prodrugs of thephosphorous-based acids thereof. Therefore, the phosphonic acid,phosphoric acid, and aminomethylenephosphonic acid are drawn andutilized interchangeably, with phosphate, phosphonate, andaminomethylenephosphonate. Biologically hydrolyzable esters of thephosphorus-based acids may also be utilized in the method of theinvention.

As used herein, the term “active concentration” refers to a quantitysufficient to achieve a desired therapeutic and/or prophylactic effect,e.g., 1) Lowers postprandial glycemic response of refined carbohydrates;2) Increases energy expenditure following ingestion of refinedcarbohydrates; and 3) Decreases appetite; and 4) Reduces body weight ofsubjects.

Definitions

The glycemic index is the extent to which blood sugar is increased uponingestion. A “reduced” glycemic index is a glycemic index value that isat least 5% lower after ingestion of an equivalently sweet amount ofsweetener in a product as described herein relative to the index of agiven amount of sucrose when ingested as refined sucrose. The reductionin glycemic index is preferably greater, e.g., 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90% or more, relative to enriched carbohydrates.

Preferably, in an enriched carbohydrate according to the invention, theflour comprises (or consists of) cereal flours. More preferably, theflour for use in a bread product according to the invention is selectedfrom the group consisting of wheat flour, whole wheat flour, complete(wheat) flour, (whole) oat flour, (whole) spelt flour, and mixturesthereof. Even more preferably, the flour for use in a bread productaccording to the invention is selected from the group consisting ofwheat flour, whole wheat flour, complete wheat flour, and mixturesthereof.

Flour—White flour contains substantially only ground endosperm of thewheat kernel, and includes very little or none of the bran, the outercovering of the grain, and the grain contained inside the kernel.

Whole wheat flour by contrast contains a reconstituted material wherethe separated bran, grain and endosperm are recombined to give a flourof high bran content. It is worth noting, however, that the amount ofbran in white flour varies from country to country. The level of bran inflour is usually quantified by measuring ash content. The ash content inwhite flour may vary between about 0.3% to around 1%. For example, inthe USA and China the ash content is usually about 0.4% and in Australiait is usually in the range of 0.5% to 0.7%. In contrast, whole mealflours in Australia have a higher ash content usually in the range of1.1% to 1.4%.

Soluble fiber—Carbohydrate which is not digestible by the humandigestive tract, but is fermented by gut bacteria.

White flour contains the ground endosperm of the grain and is a white,pure flour with the bulk of the bran or germ of the grain removed suchthat the ash content of the flour is usually less than 0.75%, preferablyless than 0.70% and even more preferably less than 0.66%. This is indistinct contrast to whole wheat flour, otherwise known as whole mealflour, which contains all of the bran and germ content of the flourgrain. The white flour used in the present invention preferably has aparticle size less than about 600 more preferably between about 90 μm toabout 500 or mixtures of flours within those ranges. Flour having aparticle size of about 500 μm is generally referred to as semolina. Thewhite flour (e.g. baker's wheat flour) used in this invention maycontain from 0 to 100% semolina, such as 10% to 100%, 20% to 100%, 30%to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to100%, 90% to 100% and 100% semolina. White flour such as standardbaker's wheat flour generally has a particle size of between about 90 μmand 180 μm, and such flour may be utilized, or mixtures thereof withsemolina in proportions already mentioned, or mixtures of white flourhaving a particle size generally from about 180 μm to about 500 μm.

If semolina is incorporated within the product it may be necessary toalter proportions of other components, for example because relative tostandard baker's wheat flour the semolina absorbs less water. In thiscase it may be appropriate to add additional non-starch polysaccharide,which increases water absorption.

Non-starch polysaccharides include one or more polysaccharides, such asa mixture of two or more non-starch polysaccharides. Non-starchpolysaccharides according to the present invention are food gradenon-starch polysaccharides, including processed forms of gums, plant andmicrobial polysaccharide extracts, polysaccharide extracts from grains(such as barley, oats), and polysaccharide extracts from seaweeds, allof which are widely commercially available and find various diverse usesin the food industry. Non-starch polysaccharides include, but are notlimited to, guar, pectins, hemicelluloses, alginates, xanthans, locustbean gum, tragacanth, psyllium, arabic, acacia, gellan, glucans such asβ-glucan, carrageenans, oat and barley fibre. Non-starch polysaccharidesare preferably added in appropriate quantity to give an amount of about0.5% to 6%, preferably 1% to 6%, more preferably 1% to 4% of a bakedwhite bread product.

The white bread product comprises a soluble fibre content of at leastabout 0.5% on a 40% water content basis, this being the total moisturecontent of the baked bread product, preferably 0.5% to 4%, morepreferably 1% to 3%, and still more preferably 1% to 2.5%. Soluble fibrecontent of the baked bread may be determined for example by the methodof AOAC 32.1.17 Official Method 991.43 (17^(th) Ed, 2000), Associationof Official Analytical Chemists, Washington D.C., USA.

Edible fats (including edible fats, oils and lipids and lipid containingor mimicking agents) are widely used in the baking industry and theseinclude vegetable oils, (including canola, soy, corn, olive, palm,coconut, and peanut), hydrogenated fats, butter, margarine, tallow,lard, eggs, marine oils (eg. fish oils), emulsifiers, fat mimics,hydrated monoglycerides and mixtures thereof. The edible fat (includingfat present in other ingredients of the product such as the flour) ispresent in the white bread product in an amount between 0 and 8%,preferably about 0.5% to about 5%, preferably 0.5% to 3% and mostpreferably greater than about 2.1%. For example, the total amount ofedible fat within the baked product can be determined by the techniqueof solvent extraction with acid hydrolysis (see AOAC Official methods,922.006, 945.44 (17^(th) Ed, first revision, 2002), Association ofOfficial Analytical Chemists, Washington D.C., USA).

Maltose content of a baked bread product, such as a baked white breadproduct, is a measure of enzymic digestion during the bread makingprocess. The white bread product according to the present invention hasa maltose content of less than about 5%, for example from about 0.5% toabout 5%, 0.5% to about 4.5%, 1% to about 3.5. A figure of about 5% orless maltose content is indicative of significant amylolytic enzymicdegradation during the bread manufacturing process. For example themaltose content of the baked product can be determined by highperformance liquid chromatography (HPLC) (see AACC Method 80-04 (10^(th)Ed, 2000), American Association of Cereal Chemistry, St. Paul, Minn.,USA or AOAC 44.4.13, Official Method, 977.20 (17^(th) Ed, secondrevision, 2003), Association of Official Analytical Chemists, WashingtonD.C., USA.).

The specific volume of a loaf of bread or bakery product is a measure ofthe density of the crumb of the bakery product, particularly a loaf ofbread. White bread is characterized by a light and fluffy crumb,associated with a high volume expanded crumb. The volume of a loaf ofbread or more properly its specific volume is measured as the volume ofthe loaf divided by weight of bread. The specific volume of the breadproduct according to the present invention is from 3 to 9 cm³/g, morepreferably between 4 to 6 cm³/g, which is characteristic of a light andfluffy high volume white bread product. Bread product specific volume isreadily calculated as: volume (cm³)/weight (g). Volume can be determinedby image analysis or seed displacement, for example.

The crumb of the baked white bread is white. That is, it has a generallywhite appearance and to the naked eye is generally visibly free frombran specks, nuts, seeds and grains. In a preferred embodiment, thecrumb of the baked bread of the invention has a CIE L*-b* value, such asmeasured using the HunterLab CIE colour scale (HunterLab, Insight onColor, Jul. 1-15, 1996, Vol. 8, No. 7), greater than 66, more preferablybetween 68 and 74; as characteristic of a demonstrably visible whitebread. Hence, the colour of the white bread product is fully consonantwith the white bread texture, taste and appearance.

The white bread product according to the invention has a lowered GI ofabout 65 or less, preferably a low GI of 55 or less, and more preferablya GI of between about 45 and about 55. For example, GI of the bread canbe determined by adopting the Standards Australia draft standard DR05435.

The low GI white bread according to the present invention retains itslow GI status when manufactured in a modern continuous baking process.

The white bread product of the present invention has significantsoftness after production, for example, with examples of the softness ofthe bread of the invention including a softness of about 0.8-2 N,preferably 1-1.2 N on one day of shelf life, and a softness of about 2-3N, preferably 2-2.5 N on five days of shelf life, following packaging ina typical polythene bag. Softness can for example be measured by forcepenetration using a Stable Microsystems Texture Analyser TaTx2 and a 36mm aluminum cylindrical probe which measures breadcrumb firmness bydetermining the force required to compress a product a specifieddistance (parameters: test speed 0.3 mm/sec, distance 10 mm and samplethickness 25 mm).

The white bread product includes but is not limited to square, liddedtin breads, unlidded breads such as high top breads, and free standingbreads such as cobs and viennas, rolls, bagels, hamburger buns,baguettes, Italian and Turkish style breads such as foccacia ciabattaand pide, and Asian style breads such as steamed buns.

The bread composition preferably contains standard suitable breadingredients, including yeast, wheat gluten, salt, soy flour, vitamins,minerals, gluten and other proteins, antioxidants, sweeteners andemulsifiers. If desired, dough conditioners (such as enzymes) and/orpreservatives can be included in the bread composition, as it will beunderstood by one of ordinary skill in the art.

The baked bread product in accordance with this invention may be made bystandard bread manufacturing means, including the conventional spongeand dough methodology, and straight dough methodology, particularlymanufactured in a standard continuous baking process. The sponge anddough method may produce breads with an improved flavour and improvedshelf-life characteristics compared to breads made by the straight doughmethod.

In the sponge and dough method, a two-stage mixing process is used.First, part of the ingredients (a portion of the total flour, yeast andwater) of a bread composition are mixed to form a “sponge” and allowedto ferment for an appropriate time, such as 3 to 4 hours at 20° C. atatmospheric or controlled humidity (such as 80% to 90% relativehumidity). After the sponge fermentation stage is completed, theremainder of the ingredients are added to the sponge, and a dough isthen formed by mixing. The dough is mixed at a suitable speed and for asuitable time to develop the dough product. Following a rest period, thedough is mechanically divided into pieces, rounded and machine molded.The molded dough pieces are placed in an appropriate container for therespective dough weight, and proved at around 25° C. to 45° C. and 70%to 90% humidity. After appropriate proving to give the appropriateheight for the given container, the containers are loaded into an ovenand baked at 200° C. to 230° C. for about 20 to 30 minutes, thisdepending on the weight of the dough and the oven type, and bread form,as would be understood by one skilled in the art. Following baking thebread is removed from the container and allowed to cool, for example for1 to 2 hours, before being bagged.

In the straight dough process, all of the ingredients for the bread aremixed into a dough in a single mixing step without formation of asponge. The dough is fermented for a suitable period of time, such asfrom about 20 minutes to about 20 hours, more preferably about 1 −4hours. The dough is then divided, weighed and processed as describedabove for the sponge and dough method.

It is possible for the dough to be chilled or frozen before baking. Suchdough can be transported from one site to another for baking or may bemade available to consumers in the form of a chilled or frozen dough,such that the consumer can then bake the bread at a convenient time tothereby enjoy fresh baked bread. The present invention encompasses thepreferably chilled or frozen dough composition and methods of producingthe dough and the baked bread product.

Two pita bread formulations viz. restored white pita bread (RWPB; torestore its pre-milling levels) and fortified white pita bread (FWPB; todouble its pre-milling levels) with macro minerals (P, K, Mg) wereprepared and their sensory properties and postprandial glycaemiacompared to those of white pita bread (WPB). The sensory characteristicsof breads were assessed using a triangle difference test and anacceptability test. Postprandial glycaemia was determined using a singleblinded cross over design whereby overnight-fasted healthy male subjectsconsumed in random order one of the 3 different types of pita bread.Palatability of pita bread was not affected by the addition ofmacro-minerals, while postprandial glycaemia of RWPB and FWPB weresignificantly lower than that of WPB at 60 minutes onwards.Additionally, RWPB and FWPB breads maintained low levels of postprandialplasma triglycerides as compared to WPB. Among the bread types, FWPBsignificantly retained the lowest GI compared to WPB. These findingsindicate that the postprandial glycaemia of whole wheat may beattributed to its content of macro minerals. At these levels ofaddition, the minerals (P, K, Mg) would have marginal effects on theproduction costs of pita bread and ingestion of about 500 g (2000 Kcal)per day would be lower than the upper limit of intake set by differenthealth agencies/organizations including institute of medicine (10M).

As used herein, a “food product” is a food in a form that does not existin nature. In embodiments, a food product includes at least two edibleingredients that do not exist together in nature. A “food” is anutritious substance that animals, including humans, pets and livestock,eat or drink. A “nutritious substance” is a macronutrient such as a fat,carbohydrate or protein, or a micronutrient such as an essential ornon-essential vitamin or mineral.

One or more phosphorous compounds described herein or derivativesthereof, alone or in combination, may be incorporated into a foodproduct. The one or more compounds may elicit a perception of saltinesswhen the food product is consumed. In embodiments, the one or morecompounds are included in a food product that contains a salt thatimparts a salty taste. Preferably, at least one of the one or morecompounds is a taste modulating compound or salty taste modulatingcompound.

In embodiments, a food product includes an ingredient, a salt thatimparts a salty taste, and a taste modulating or salty taste modulatingcompound. The ingredient may be a nutritious ingredient; that is, aningredient that is a nutritious substance. The taste modulating or saltytaste modulating compound may be present in the food product in anamount sufficient to enhance the salty taste of the food product. Inembodiments, the ingredient, the salt and the taste modulating or saltytaste modulating compound are present in the food product in amounts orconcentrations not found in naturally existing food products, such asbananas, peppers, avocadoes, wheat, or the like.

Hereinafter, the present invention is more specifically described by wayof examples; however, the present invention is by no means limitedthereto, and various applications are possible without departing fromthe technical idea of the present invention.

Examples Example 1: Phosphorus Ingestion Improves Oral Glucose Toleranceof Healthy Male Subjects

The example was comprised of two experiments. Healthy male subjects wererecruited to perform the experiments (Table 1). Placebo (cellulose) or P(125 mg of potassium phosphate per tablet) tablets, which had similarweight and color, were administered to subjects in a randomized order toprevent order-of-treatment effect. The consent forms were obtained frompatients.

TABLE 1 Characteristics of the study subjects Characteristics Mean ± SEMExperiment 1 (n = 7) Age (years) 23.22 ± 1.83 Weight (kg) 68.88 ± 4.05Height (m)  1.74 ± 0.02 BMI (kg/m²) 22.65 ± 0.82 Fasting glucose (mg/dl)86.38 ± 1.62 Fasting triglycerides (mg/dl) 90.63 ± 15.9 Fastingphosphorus (mg/dl)  4.06 ± 0.19 Experiment 2 (n = 8) Age (years)  27.3 ±1.68 Weight (kg)   73 ± 4.78 Height (m)  1.76 ± 0.04 BMI (kg/m²)  23.5 ±0.97 Fasting glucose (mg/dl)   88 ± 2.17 Fasting triglycerides (mg/dl) 119 ± 15.3 Fasting phosphorus (mg/dl) 3.75 ± 0.2

Experiment 1: The Effect of Phosphorus Ingestion on Oral GlucoseTolerance Test [OGTT]

Seven overnight fasted subjects (age (mean±SEM): 23.2±1.83 years; BMI:22.65±0.82 kg/m2) (Table 1: Experiment 1) were asked to attend 3experimental sessions that were separated by a minimum of 3 days.Sessions included the consumption of either 500 mg of P (4 tablets), aglucose solution (75 g glucose) with 4 Placebo tablets, or a glucosesolution (75 g glucose) with 500 mg P. All were ingested with 250 ml ofcold water. Blood was withdrawn at base-line and monitored till 240 min(min) after consumption.

Experiment 2: The Effect of Pre-Phosphorus Ingestion on OGTT

Based on data from experiment 1, peak serum P concentration (60 min) wasassociated with significant decrease in serum glucose and insulinlevels. Thus, experiment 2 was designed to investigate whether P intakeone hour prior to glucose ingestion would potentiate the effect of P onpostprandial glucose and insulin levels. Eight over-night fastedsubjects (age: 27.3±1.68 years; BMI: 23.5±0.97 kg/m²) (Table 1:Experiment 2) attended 2 experimental sessions that were separated by aminimum of 3 days. Subjects were given placebo or P (500 mg) tablets 60min prior to glucose ingestion. Blood was drawn at baseline (−60 min)and monitored till 240 min relative to glucose ingestion.

Serum was separated from collected blood samples and stored at −80° C.for later analysis of glucose, total P, tri-glyceride and insulin.Glucose was measured from venous samples. Insulin sensitivity wasestimated by the method of Caumo A, Bergman R N, Cobelli C. Insulinsensitivity from meal tolerance tests in normal subjects: a minimalmodel index. J Clin. Endocrinol. Metab. 2000; 85:4396-402 and expressedas (×10⁴ dl·kg⁻¹·min⁻¹·μUml⁻¹). The method is based on the kinetic ofboth glucose and insulin through coupling their rate of appearance intocirculation following oral glucose ingestion. It is based on simple areaunder the curve type of calculation and was validated in normal subjectsin whom their calculated insulin sensitivity was strongly correlated tothat of frequently formula sampled IV glucose test (FSIGT). In addition,index of insulin sensitivity was calculated using the composite equationproposed by Matsuda M, DeFronzo R A. Insulin sensitivity indicesobtained from oral glucose tolerance testing: comparison with theeuglycemic insulin clamp. Diabetes Care. 1999; 22 (9): 1462-7.

Data are presented as means±SEM. Paired t-tests were used to comparebetween treatments and to detect the difference from baseline withineach treatment. Repeated Measure ANOVA was run to test the effect oftreatment groups over time on each of the dependent variables (P,Glucose and insulin). The level of significance was fixed at P<0.05.

Baseline serum levels of the different parameters (total P, glucose,insulin) were similar between sessions and both experiments (Table 1).

Ingestion of P alone increased serum P significantly, while ingestion ofglucose alone decreased postprandial serum P levels. The pattern ofserum P changes following glucose and P ingestion (G+P) was differentthan that of the other two treatments (FIG. 1a ), in line, repeatedmeasures ANOVA showed that serum P was significant according totreatment (Table 2: Experiment 1).

TABLE 2 Repeated measure ANOVA outcome variables (Phosphorus, glucoseand Insulin) of the two experiments Time Treatment Interaction Outcomevariables (P value) (P value) (P value) Experiment 1 (n = 7)^(a) SerumPhosphorus 0.616 0.000 0.321 Serum Glucose 0.000 0.145 0.207 SerumInsulin 0.000 0.168 0.619 Experiment 2 (n = 8) Serum Phosphorus 0.0010.002 0.498 Serum Glucose 0.000 0.815 0.961 Serum Insulin 0.000 0.2330.724 ^(a)Only glucose with placebo and glucose with P groups wereincluded in the analysis.

Postprandial serum glucose concentration of the P treatment groupsignificantly decreased by around 5 mg/dl during the experimentalsession, but this minimal reduction is believed to be the result offasting. Glucose ingestion increased postprandial serum glucose levelsof both treatments, glucose and G+P, but the magnitude of the increasewas significantly lower in the G+P as compared to glucose treatment attime 60 min (P=0.016), (FIG. 1b ). Repeated measures ANOVA showed thatserum glucose levels were significantly different according to time, butfailed to reach statistical significance between treatments (Table 2:Experiment 1).

Ingestion of P alone did not alter postprandial insulin concentration.Insulin levels of the G+P treatment were significantly lower (P=0.002)than that of the glucose treatment at time 60 min (FIG. 1c ). Repeatedmeasures ANOVA found that serum insulin was significant according totime only (Table 2: Experiment 1). Insulin sensitivity obtained fromoral glucose tolerance test and according to Caumo et al. formulaincreased following G+P treatment and the difference was close tosignificance (P=0.051) (Table 3: Experiment 1). While index of insulinsensitivity increased significantly (P=0.006) following the addition ofP to OGTT (Table 3: Experiment 1). Insulin sensitivity of the Pingestion treatment was not determined since postprandial glucose andinsulin levels were minimally affected.

TABLE 3 Measures of insulin sensitivity from oral glucose tolerance testPaired t-test Outcome variables Placebo Phosphorus (P value) Experiment1 (n = 7) Insulin sensitivity index [30]  5.69 ± 0.86  7.00 ± 1.06 0.006Insulin sensitivity [29] 12.19 ± 3.85 20.22 ± 6.65 0.051 Experiment 2 (n= 8) Insulin sensitivity index [30]  9.17 ± 0.97  8.88 ± 0.78 0.633Insulin sensitivity [29] 15.31 ± 2.58 18.39 ± 3.28 0.210

Ingestion of placebo tablets had no effect on serum P levels prior toglucose ingestion, but glucose ingestion decreased serum P levels (FIG.2a ). Following P ingestion, serum P levels increased significantly attime 0 and 15 min and then returned to baseline levels (FIG. 2a ).Repeated measures ANOVA analysis of all time points showed that serum Pwas significant according to time and treatment (Table 2: Experiment 2).Serum glucose levels of the glucose and G+P treatments increasedsignificantly following glucose ingestion (FIG. 2b ). The increase inglucose levels was similar between the two treatments, except for aslight difference at time baseline and 240 min believed to be of noclinical significance (FIG. 2b ). In line, repeated measures ANOVA weresignificant according to time only (Table 2: Experiment 2).

Glucose ingestion increased insulin levels of both treatments, but themagnitude of this increase was modestly lower in the P preload treatedgroup (FIG. 2c ) and repeated measures ANOVA failed to detect differenceaccording to treatment (Table 2: Experiment 2). Measures of insulinsensitivity were found to be similar between the two treatments and thiswas expected since the changes in insulin and glucose were similarbetween the treatments (Table 2: Experiment 2).

DISCUSSION

In agreement with other studies, ingestion of P alone (experiment 1 and2) increased postprandial serum P, while ingestion of grape juice,glucose alone, or other type of carbohydrate reduced postprandial serumP levels. Glucose may indirectly affect P status through the stimulationof peripheral P uptake by insulin, which in turn stimulates thephosphorylation of several compounds including carbohydrate, fat andprotein. About 60% of in-fused P has been reported to be translocatedfrom the extracellular to the intracellular compartment, mainly inskeletal muscles. This translocation is believed to be mediated viainsulin action as glucose infusion in pancreatectomic dogs failed toinduce a reduction in serum P except when insulin became available. Inline with this, the parallel increase in postprandial glucose andinsulin (peaks at 30-60 min) was followed by a decrease in serum P (dipat 90 min). Therefore, under conditions of P intake alone, intracellularP is likely to have been affected since insulin was not altered.

In the current example, both co- and pre-ingestion of P were able tohalt the drop in serum P levels following glucose ingestion. In the G+Ptreatment, increased P uptake and availability might have contributed tothe drop in glucose and insulin levels at time 60 min, due to anincrease in intracellular glucose trapping (phosphorylation), especiallysince insulin release depends on glucose circulation. This process mighthave played a role in the observed improvement in the measures ofinsulin sensitivity following P ingestion with glucose (Experiment 1).Phosphorylation or P trapping seems to have been substantiallystimulated or became dependent on extracellular P 30 min after glucoseingestion; as indicated by the drop in postprandial serum P levels. Thismay partially be behind the failure of P preingestion to impactpostprandial plasma glucose, since the majority of phosphorus is knownto be absorbed within 60 min, as supported by the finding fromexperiment 1.

When glucose was ingested alone, the low availability of P may havehindered insulin phosphorylation capacity through creating competitionfor P. Such competition may have deleterious effects since it can affectglucose clearance and trapping, glycolysis and gluconeogenesis, andphospholipids and hepatic fat accumulation. Therefore, postprandialglycemia and insulinemia seem to be improved by exogenous P availabilityand this may partially explain the reported association between lowserum P with insulin resistance and elevated blood glucose levels.

Ingestion of P before or with glucose was able to prevent the drop inpostprandial serum P levels. The sustained high postprandial serum P inthe G+P treatment in comparison to that of the glucose ingestion aloneimplies that intracellular P uptake may be controlled by a limitedcapacity for phosphorylation and/or glucose up-take. In healthysubjects, peripheral glucose uptake, especially in skeletal muscles, isknown to be triggered by insulin dependent Glut 4 stimulation. While,intracellular glucose phosphorylation is controlled by the activities ofglucokinase (liver) and hexokinase (muscle), the latter has a low Vmax(maximum velocity) capacity and is highly inhibited by glucose-Pproduction. The reduction in serum glucose of the G+P treatment arguesagainst a defect in Glut 4 (glucose uptake), therefore the sustenance inplasma P may have been attributed to the low Vmax capacity of musclehexokinase. Accordingly, ingestion of higher P doses would not beexpected to further improve glucose, insulin, or insulin sensitivity.

The fact that P is absorbed along the entire intestinal tract could beresponsible for the observed high plasma P levels (above baseline value)in the G+P treatment beyond the time (120 min) of availability ofglucose and insulin. Moreover, the difference in the magnitude ofchanges in postprandial glucose and insulin levels between the preloadand the co-ingestion experiments implies that factors beyond theavailability of circulating P, glucose and insulin may have beeninvolved in improving of insulin sensitivity. The weak significantassociation seen between P preload on insulin sensitivity could beexplained by the small sample size of the present example. On the otherhand, glucose-phosphorus interaction in the proximal part of the smallintestine may been involved in insulin sensitivity through incretinhormones. These hormones, especially glucagon like peptide-1 (GLP-1) andgastric inhibitory polypeptide (GIP) are known to be secreted inresponse to meal ingestion, especially high protein meals (rich in P)and were reported to affect insulin status and to play an important rolein regulating postprandial blood glucose.

The observed improvement in the measures of insulin sensitivityfollowing meal-phosphorus co-ingestion may have been partially involvedin the reported synergic relationship between the intake of whole grainsand glucose tolerance. Especially since this relationship was notexplained by the function of dietary fiber and whole grains are rich inphosphors. Additionally, this observation may partially explain theobserved parallel rise in metabolic syndrome with global urbanizationand westernization of dietary habits, which favor low P intake. Incomparison to other studies in the literature, which have used Pinjections to study its effect on glycaemia and insulinemia, the currentexample used a different method that mimicked daily dietary habits,through the ingestion of 500 mg of P with a glucose load (approximately1.7 mg of P per Kcal). Therefore, these findings highlight the role of Pin improving the states of hyperglycemia and hyperinsulinemia in healthyindividuals without the influence of serum calcium and FGF-23 which didnot vary when P is used.

On the other hand, elevated fasting serum P levels were reported to beassociated with mortality among patients with chronic kidney andcoronary diseases. The association was partially explained by thecapacity of high P conditions to induce vascular calcification andendothelial injury using in vitro studies. Moreover, in a human (invivo) study endothelial function impairment was apparent under high(1200 mg P/meal) and not normal (400 mg P/meal) P ingestion. In fact,the negligible impact of dietary P intake on serum P levels implies thatfactors associated with increased serum P, rather than P intake, wereprobably behind the association between cardiovascular disease and serumP. Recently, a weak association between dietary P intake and all-causemortality was reported and this was questioned since participantsadopted different dietary patterns and P intake was not the onlyvariable. Thus, the nature of the relation between P intake andcardiovascular disease and mortality is far from clear and requiresfurther scrutiny.

In the present example, sample size was based on the previously reporteddata of AUC for glucose. However, the observed large variations betweensubjects seem to have diluted the impact of the interventions. Furtherstudies, using a larger sample size, would help in exploring themechanisms by which the observed effects are mediated especially byexamining the role of incretin hormones.

Although the dietary habits of these subjects were not assessed prior tothe initiation of the study; however, as stated previously, fastingserum P status is not a good indicator of P intake. Postprandial statusof the measured parameters is not likely to be affected by prior mealintake since all subjects were overnight fasted. Therefore, assessingdietary habits of subjects has no added implications on the exampleresults.

Example 2: Increased Phosphorus Content of Preload Suppresses Ad LibitumEnergy Intake at Subsequent Meal

A preliminary set of experiments was conducted to investigate the effectof increased phosphorus content of a preload solution on subsequent foodintake. The effect of water, sucrose (50 g), fructose (40 g fructoseplus 10 g glucose) or glucose (50 g) preloads was examined with orwithout the addition of 500 mg of phosphorus (mixture of potassium andsodium phosphate). This work was approved by the Institutional ReviewBoard of the American University of Beirut and written informed consentwas obtained from all subjects. In brief, participants who had stablebody weight for the last 3 months and were unrest-rained eaters asassessed by the three-factor eating questionnaire were selected. Allsubjects were regular breakfast consumers and were asked to maintaintheir regular dietary and physical activity habits throughout the courseof the example. Subjects were asked to avoid alcohol consumption, aswell as any unusual strenuous exercise 24 h before the example. After aminimum of a 12-h fast, they consumed a standard breakfast (440 kcal) 4h before presenting for the example. Participants chose a time between1100 and 1400 to be convenient to perform the test; thereafter, theywere asked to arrive at the same time on the same day of the week forthe second study session. Thus, each experiment used a within-subjectdesign, wherein each participant served as his or her own control.

In each experiment, two chilled preloads (with or without addedphosphorus) were offered in a blind randomized order so as to controlfor the order-of-treatment effect. Each subject presented for two studydays separated by a minimum of 1 week. All preloads were flavored withlemon to mask the taste of added phosphorus, had a volume of 250 ml andan additional 150 ml of pure water was offered to each subject (to washout any after-taste); thus, a total of 400 ml of liquid solutions weredrunk on each study day. At 80 min after the preload, an ad libitumlunch, consisting of standard pizza and water, was offered. Subjectswere asked to eat freely until they felt ‘comfortably full’; both foodand water intakes were measured. Water intake was measured by weight (g)and food or energy intake (kcal) was obtained from the overall weight ofpizza consumed.

In all experiments, the addition of phosphorus to the different preloadswas associated with a significant reduction in energy intake at thesubsequent meal (Table 4).

TABLE 4 Characteristics of subjects and ad libitum energy intakefollowing the ingestion of phosphorus manipulated preloads. (ad libitum)at Gender Age BMI Energy subsequent Preload (n) (years) (kg m-2) intakemeal −P +P Water M (12) 23.8 ± 4.4 23.4 ± 3.0 1534 ± 341 1119 ± 210* Sucrose M (5), 21.7 ± 4.0 22.2 ± 1.3  770 ± 379 532 ± 341* F (5) Fruc- M(10), 26.6 ± 5.5 27.2 ± 1.4 1012 ± 407 676 ± 404* tose F

Glucose M (11) 20.7 ± 1.4 24.7 ± 1.2  890 ± 308  652 ± 291**Abbreviations: BMI, body mass index; F, female; M, male. (−P): no addedphosphorus to preload. (+P): with 500 mg of added phosphorus to preload.Results are mean ± s.d. *P < 0.001, **P < 0.01, paired t-test (−P) vs(+P).

indicates data missing or illegible when filed

The magnitude of reduction varied slightly. The addition of phosphorusto the water preload led to 27% reduction in energy intake at thesubsequent meal, similar to that of glucose (25%), while the addition ofphosphorus to the sucrose or fructose preloads led to 33 and 35%reduction in energy intake at the subsequent meal, respectively. It isworth noting that phosphorus addition to the different preloads failedto affect water intake (data not shown), which indicates that thesuppression of food intake was not due to osmotic differences caused bythe presence or absence of phosphate. Data from the water preloadexperiment indicate that the reduction in subsequent energy intake isnot dependent on carbohydrate energy content of the preload.

These findings are in support of the hypothesis that phosphorus contentof a preload reduces subsequent food intake, although the exactmechanism by which this occurs was not investigated. What also remainsto be studied is whether such an effect is influenced by body weight,and whether it persists in obese subjects.

The scope of the present study did not allow the scrutiny of centraland/or peripheral factors. However, the present study provides humandata wherein the stimulation of food intake and reduction in hepatic ATPstatus of rats after two 5 AM injections were reversed by pretreatmentwith a phosphorus infusion. Thus, it is prudent to assume that thenegative association between phosphorus content of the preload andsubsequent energy intake is related to hepatic ATP status, which isknown to be attenuated by phosphate preloading. Such an assumption is inline with other human studies that have shown that hepatic ATP store andfractional recovery are inversely related to body mass index, and serumPi is negatively related to body weight. Moreover, an analysis ofmetabolic data using the Knowledge Discovery in Databases procedureconcluded that ATP deficiency or decreased energy levels were stronglylinked to the development and sustenance of obesity, by drivingovereating and conserving energy.

Hepatic ATP status is known to be influenced by both phosphorus contentof a meal and hepatic phosphorylation of metabolites, and thus whatstill remains to be determined is whether phosphorus content of thesecond meal contributed to the observed reduction in energy intake. Atthe same time, it is not clear whether the effect of phosphorus contentof preload on subsequent energy intake can be extended beyond the 80-minperiod used in this experiment. The present finding may in part help toclarify the existing controversy over the relationship between dairyproduct (high in phosphorus) consumption, calcium and body composition.In addition, these findings raise the possibility that the reducedenergy intake observed in high-protein diets may partially be explainedby the high phosphorus content of protein meals. These findings may alsohave implications for the consumption of refined cereal products,because cereals, particularly wheat, lose most of their phosphoruscontent during the refining process. This may call for the enrichment ofrefined cereals with phosphorus in an attempt to reduce energy intake.

Example 3: Increased Postprandial Energy Expenditure Following theAddition of Phosphorus to a High Carbohydrate Meal

The objective was to determine the effect of phosphorus (P) ingestionwith high carbohydrate meal on postprandial energy expenditure. Pingestion increases postprandial thermogenesis of the subjects.

A cross over study was conducted on six lean male subjects. Subjectsreceived a 500 Kcal high carbohydrate meal (CHO: 65% E; Fat: 31% E;Protein: 4% E) with (500 mg of P) or without P. Energy expenditure wasmeasured at baseline and at 30 minute intervals for 4 hours followingmeal ingestion using a ventilated hood and canopy system COSMED QUARKCPET unit.

FIGS. 3A-3C shows the postprandial energy expenditure of meal containingP was significantly higher than that of placebo (p=0.007). This increasewas associated with a significant rise in fat oxidation (%) (p=0.022),while carbohydrate oxidation (%) was decreased (p=0.023).

P was able to increase postprandial energy expenditure mainly due toincreased fat oxidation. This data has promising effect for themanagement of obesity.

Example 4: Effect of Phosphorus Supplementation on Weight Gain and WaistCircumference of Overweight/Obese Adults

Participants: After approval of the study by the institutional reviewboard at the American University of Beirut (Beirut, Lebanon), 63 adultsaged 18 to 45 years with a BMI≧25 kgm-2, who provided signed informedconsent, were recruited from the general public using posteradvertisements or direct approach. Details about recruitment,randomization and follow-up are presented in FIG. 4. Exclusion criteriaincluded glomerular filtration rate of 60 ml min-1 per 1.73 m², presenceof any significant medical disease, pregnancy or lactation, regularadministration of drugs that affect body weight and weight change of ≧3%within 3 months before the study. The enrollment of 40 subjects (20 pergroup) would detect a 10% change in weight of the placebo group,assuming the latter having a mean weight of 90 kg and s.d. of 10 kg,with 80% power and an α of 5%.

Randomization and Masking

This double-blind, randomized, controlled study allocated subjects intoplacebo group (n=21) or phosphorus group (n=26). Participants wererequested to take three tablets containing either 375 mg phosphorus or aplacebo (Nutricap Labs, Farmingdale, N.Y., USA) with each main meal(breakfast, lunch and dinner) for 12 weeks. They were asked to maintainregular dietary and physical activity habits during the entire studycourse and avoid alcohol consumption and any strenuous exercise 24 hbefore their visits (at baseline, 6 weeks and 12 weeks). Assignment tointervention or control group was made by having the principleinvestigator (corresponding author) ask the eligible subjects to blindlydraw an envelope from a large box of 100 opaque, sealed envelopes (50for each group), each containing a 2-cm by 2-cm paper with a writtencode designating intervention or control. There were no detectabledifferences in size or weight between intervention and controlenvelopes. In addition, both researchers and participants were blindedfor the type of supplements that were similar in size, shape, color andodor.

Procedures

Subjects were asked to attend the research unit at baseline and after 6and 12 weeks of participation. At baseline, anthropometric measurementsand blood samples were collected and a subjective appetite questionnairebased on Wilson et al. Appetite assessment: simple appetitequestionnaire predicts weight loss in community-dwelling adults andnursing home residents. AJCN 2005; 82:1074-1081 was completed.Participants were given a 6-week supply of the allocated supplement andwere asked to attend the research unit at the end of this period. At 6weeks, remaining tablets were collected and counted in order to assessadherence to the allocated intervention. Participants were then given asupply of the same type of supplementation for the consequent 6 weeksand were asked to complete the subjective appetite questionnaire. At 12weeks, data were collected similar to the baseline visit, and remainingtablets were counted to assess compliance. Individuals who consumed 470%of the allocated tablets were excluded. Body weight and height (withoutshoes) were measured to the nearest 0.1 kg and 0.1 cm, using acalibrated Seca balance (Hamburg, Germany) and a portable stadiometer,respectively. Blood was withdrawn after overnight fast and samples werecentrifuged for 15 min at 3500 r.p.m. at 3° C. for serum and plasmaseparation. Sample aliquots were stored at −80° C. until analysis. Serumphosphorus, creatinine, C-reactive protein, total cholesterol,high-density lipoprotein cholesterol, triglyceride and glucose levelswere measured using the Vitros 350 analyzer (Ortho Clinical Diagnostics,Johnson and Johnson, Buckinghamshire, UK). The Friedwald formula wasused to calculate low-density lipoprotein cholesterol levels. FriedewaldW T, Levy R I, Fredrickson D S. Estimation of the concentration oflow-density lipoprotein cholesterol in plasma, without use of thepreparative ultracentrifuge. Clin Chem 1972; 18: 499-502. Fastinginsulin concentration was measured using the ELISA kit (DiametraMillipore, Billerica, Mass., USA). HOMA-IR (homeostasis model assessmentof insulin resistance) was calculated as described by Matthews et al.Homeostasis model assessment: insulin resistance and β-cell functionfrom fasting plasma glucose and insulin concentrations in man.Diabetologia 1985; 28: 412-419. Glomerular filtration rate wascalculated using CKD-EPI (Chronic Kidney Disease EpidemiologyCollaboration) estimated glomerular filtration rate.

Statistical Analysis

Pairwise changes from baseline to 12-week follow-up anthropometric andbiochemical variables were tested using paired t-tests, and intergroupassessment was performed using two-sample t-test. Repeated measuresanalysis of variance test was applied to analyze intragroup variation ofappetite scores at different periods of time (baseline, 6 weeks and 12weeks). Statistical analyses were conducted using SPSS 22 (Chicago,Ill., USA).

Abbreviations: BMI, body mass index; CRP, C-reactive protein; GFR,glomerular filtration rate; HDL-C, high-density lipoprotein cholesterol;HOMA-IR, homeostatic model assessment of insulin resistance; LDL-C,low-density lipoprotein cholesterol. SI conversion factor: to convertserum phosphorus to mmol 1-1, multiply by 0.323; cholesterol, LDL-C andHDL-C to mmol 1-1, multiply by 0.0259; triglycerides to mmol 1-1,multiply by 0.0113; glucose to mmol 1-1, multiply by 0.0555. Calculatedas weight in kg divided by height in m squared. Measured at the midpointbetween the lower rib and iliac crest. Because of missing data, based onsample size of 20 and 26 for placebo and phosphorus groups,respectively. Because of missing data, based on sample size of 20 and 25for placebo and phosphorus groups, respectively.

Subject Characteristics

Baseline characteristics are shown in FIG. 5, and they were similarbetween groups. In all, 47 participants (placebo group n=21; phosphorusgroup n=26) completed the intervention, and all subjects had normalglomerular filtration rate (460 ml min-1 per 1.73 m²) with a mean of114.14 (10.19) ml min-1 per 1.73 m² and 112.24 (13.46) ml min-1 per 1.73m² for the placebo and phosphorus groups, respectively. The mean age was36.67 (9.76) years in the placebo group and 34.04 (11.24) years in thephosphorus group. No side effects were reported by participants over theexperimental period.

Anthropometric Assessments

Changes in anthropometric and biochemical characteristics from baselineto 12 weeks are shown in FIG. 6. Body weight of the placebo groupincreased significantly from baseline by 1.13 kg (95% confidenceinterval (CI) 0.19 to 2.06), whereas that of the phosphorus groupdecreased by 0.65 kg (95% CI −1.69 to 0.40). These variations resultedin a significant difference (P=0.01) in the changes in body weightbetween the two groups. Consequently, the changes in BMI of the placebogroup (0.42 kgm-2, 95% CI 0.05 to 0.78) was significantly different(P=0.01) than that of the phosphorus group (−0.24 kgm-2, 95% CI −0.59 to0.12). Simultaneously, waist circumference of the phosphorus group wassignificantly reduced by 3.62 cm (95% CI −4.90 to −2.33), and thisreduction was significantly different (Po0.001) from the small increaseof 0.38 cm (95% CI −0.44 to 1.20) in the waist circumference of theplacebo group.

Biochemical Assessments

Placebo or phosphorus treatment for 12 weeks did not affect serum levelsof phosphorus, total cholesterol, low-density lipoprotein cholesterol,high-density lipoprotein cholesterol, triglyceride, glucose andC-reactive protein. Serum levels of insulin and HOMA-IR were similarbetween the two treatments at baseline and at 12 weeks, although a mildbut significant difference was detected in their changes. This mildchange is not believed to be of clinical significance (FIG. 6).

Subjective Appetite Scores

Baseline subjective appetite scores were similar between groups. Thechanges in several parameters of subjective appetite scores were foundto decrease as the experiment progressed including that of appetite,quantity of food to reach fullness, hunger and number of snacks.However, changes in appetite, quantity of food to reach fullness, tasteof food and number of snacks was significantly reduced in the phosphorusgroup as compared with the placebo as shown in FIG. 7.

Several dietary patterns and interventions were reported to improve bodyweight. High protein diets were constantly found to induce weight loss,probably because of their capacity to decrease energy intake andincrease energy expenditure. Consumption of dairy products was alsoshown to be inversely related to body weight, whereby its increasedintake among overweight individuals was reported to lower body weight,irrespective of its calcium content. Moreover, the intake of wholegrains was shown to be negatively associated with the risk of differentcomponents of the metabolic syndrome, including body weight; however,the mechanism of such effect remains uncertain. This raises thequestions on the role of macronutrients in weight reduction, especiallyas these dietary patterns or interventions have varied macronutrientprofiles. The common feature between these diets seems to be theirphosphorus content, as proteins, dairy products and whole grains arerich sources of phosphorus. This rationale for the involvement of lowphosphorus status in the development of obesity and metabolic syndrome.

This study found that the ingestion of 375 mg phosphorus with each mainmeal, over a period of 12 weeks, was able to prevent weight gain and toreduce waist circumference among overweight and obese adults. However,minimal alterations were observed in the measured biochemical parameters(lipid profile, glucose and so on) that may be attributed to the modestbaseline abnormalities in these parameters, short experimental durationand/or to the modest anthropometric changes. The absence of change infasting plasma phosphorus further confirms that it is not a good markerof phosphorus intake.

The anthropometric changes in the phosphorus group are in line withother studies in which phosphorus status was reported to be inverselyrelated to body weight and waist circumference. The mechanism(s) bywhich phosphorus affected body weight may have been related to itsinvolvement in food intake control and/or energy metabolism. Phosphorusavailability is known to stimulate ATP production, in particular hepaticATP that is believed to transmit afferent neural signals to the centralnervous system resulting in a decrease in food intake through thestimulation of satiation. Such effect was believed to be behind theimpact of phosphorus addition to different carbohydrate preloads on thesuppression of ad libitum energy intake at subsequent meal. Inagreement, as reported in the subjective appetite questionnaires,satiation indicated by the quantity of food to reach fullness wasreduced in the phosphorus group; however, the number of main meals,which is an indicator of satiety, was not reduced. Sustenance of hepaticATP production over the postprandial and postabsorptive periods may havecontributed to the observed reduction in appetite and number of snacksand these may have been translated by subjects into taste changes.Conversely, the similarity in the scores of hunger (that is,physiological controlled by depletion of energy stores) and the numberof main meals between the phosphorus and placebo groups may be explainedby a limited availability of hepatic ATP substrates beyond postprandialand postabsorptive periods, and thus an inability to impact theinitiation of the next main meal. In brief, the impact of phosphorussupplement on energy intake seems to be related to its capacity toreduce the size of main meals (low appetite and high fullness) as wellas intake between meals (number of snacks).

Furthermore, the favorable differences in body weight and waistcircumference in the phosphorus group may have been partially related toan effect of phosphorus on energy metabolism. The addition of phosphorusto orange juice was reported to increase postprandial thermogenesisamong obese but not lean subjects. In addition, phosphorussupplementation in a weight reducing program was found to increaseresting metabolic rate of obese subjects. The pronounced reduction inwaist circumference in the face of the modest reduction in body weightmay have been attributed to changes in body composition. Weight gainunder phosphorus-deficient diet was reported to be largely attributed toan increase in adipose tissue, whereas nitrogen retention was impaired,and this seems to mimic that of low-protein (low-phosphorus) diet.Changes in body fat were reported to be related to energy intake,whereas changes in lean body mass were related to the intake of protein,known to be high in phosphorus and this raises a question of whether theeffect of protein on weight gain is linked to its content of phosphorus.It is not clear whether phosphorus supplementation favored lean bodymass retention that ultimately masked the effect on changes in bodyweight because of its capacity to retain water. In any case, theobserved reduction in waist circumference was similar to that reportedin subjects under low-fat diets, and is believed to be of clinicalsignificance as it is an indicator of abdominal obesity (visceral fat)that is known to be a risk factor of type 2 diabetes and cardiovasculardisease.

Many concerns were raised on the relation between phosphorus status andcardiovascular disease and mortality, although the nature of therelation with phosphorus intake is far from clear and requires furtherscrutiny, especially as fasting serum phosphorus does not reflect intakeas confirmed by the results. The fact that fasting but not non-fasting(that reflects intake rather than clearance) serum phosphorus levelswere associated with increased mortality and fasting serum phosphoruslevel but not dietary intake were associated with coronary arterycalcification may imply that factors behind or associated with elevatedfasting serum phosphorus rather than phosphorus intake may haveattributed to these detrimental effects. The recent reported weakassociation between dietary phosphorus intake and all-cause mortalitywas questioned as varied dietary habits or profiles were seen among thedifferent dietary phosphorus intake quartiles. Moreover, suchassociation may have been cofounded by the source of phosphorus in thediet, especially as dietary heme iron intake (derived from animal sourcethat is also high in phosphorus) was shown to increase the risk ofcardiovascular disease. It is believed that the need of phosphorusespecially for carbohydrate metabolism may have been compromised bymodernization (refinement and so on), particularly in staplecarbohydrate-rich foods (rice, wheat and so on). The impact of such acompromise is expected to depend on the contribution of staple food tototal energy intake and may partially be behind the drastic increase inobesity in developing countries, in particular as carbohydratecontribution to total energy intake is inversely related to income.

The strength of the study was that a rigorous system of training andcertification of study personnel was developed and implemented forcollecting all data. In addition, this study is pragmatic, randomized,double blinded and placebo controlled that required the use of tabletswithout requesting behavioral or dietary changes to avoid the problem ofadherence.

Example 5: Effect of Macro-Mineral Supplementation on Sensory Propertiesand Postprandial Glycaemia of White Pita Bread

Materials and Methods

This study was conducted according to the guidelines laid down in theDeclaration of Helsinki and all procedures involving humansubjects/patients were approved by the Institutional Review Board (IRB)committee at the American University of Beirut (AUB). Written informedconsent was obtained from all subjects. The clinical trial wasregistered with Clinical Trial.gov, NCT02598986.https://register.clinicaltrials.gov Wheat flour (80% extraction;Bakalian Flour Mills, Beirut, Lebanon) was used and levels of mineralsupplementation were made. Restoration, whereby the level of addedminerals to white flour aimed at attaining back original levels (priorto processing and milling), whereby each Kg of white flour contained 3.6g MgCO₃ and 12.5 g KH₂PO₄. Fortification, whereby the level of addedminerals to white flour was almost double that of original levels andeach Kg of white flour contained 7.2 g MgCO₃ and 25 g KH₂PO₄. Theamounts of added P and Mg are considered safe, since both are lower thanthe tolerable upper limits set at 4,000 mg/day and 350 mg/day for P andMg, respectively. After supplementation, different types of pita breadwere made and used for the different tests.

Pita Bread Making

Bread samples were prepared as previously in Toufeili I, Ismail B,Shadarevian S, et al. (1999) Role of Gluten Proteins in the Baking ofArabic Bread. J Cereal Sci 30 (3), 255-265. Dough contained flour (100parts), sugar (2 parts), salt (1.6 parts), yeast (1 part) and water (57parts). The ingredients were mixed (DITO SAMA, Model BM 20S, France) atlow speed for 7 min until a smooth continuous dough was obtained. Thedough was incubated at 40° C. for 15 min and the fermented dough wasthen divided into balls (˜30 g) and proofed at 40° C. for 30 min. Theballs were flattened into sheets, 1.5 mm thick, proofed at 40° C. for 15min, and baked at 500° C. to optimum crust color. The bread loaves wereplaced in polyethylene bags and stored at −10° C.

Upon termination of the bread making process, the three bread types werethen analysed for their mineral content by inductively coupled plasmamass spectrometry (ICP-MS) using the standard method EPA 200-7/8, (1991)Methods for the determination of metals in environmental samples.

Experiment 1: Difference and Acceptability Sensory Tests

Twenty four healthy untrained male volunteers participated in adifference/discrimination test. Two triangle tests (1 set of threesamples at a time) were conducted to compare white pita bread (WP) vs.restored (WP-R) or vs fortified (WP-F) pita bread. Panelists were askedto indicate the odd sample in each set and to rinse their mouths beforeeach sample. A consumer acceptability test was conducted with 60 healthyrandomly recruited panelists (29 females and 31 males, mean age 22years) from AUB as described in Lteif et al. The characterization of thephysicochemical and sensory properties of full-fat, reduced-fat andlow-fat ovine and bovine Halloumi Cheese. J Dairy Sci 92, 4135-4145(2009). The three samples used in difference tests were assessed. Tengrams of each type of pita bread were prepared 2 hours prior to servingthem and were stored in the refrigerator (4° C.). Panelists ratedoverall acceptability, appearance, colour, odour, flavour and texture ona 9-point hedonic scale. Lawless H T & Heymann H (2010) SensoryEvaluation of Food. Springer. All products were served in 59-mL plasticcontainers in individual booths. Panelists were instructed to rinsetheir mouths before each sample and the order of the samples within eachset was randomized among the panelists in both tests.

Experiment 2: Determination of Postprandial Glycaemia

Twelve healthy males were randomly recruited and were asked to maintaintheir regular dietary and physical activity habits during the entirestudy course, and to avoid alcohol consumption as well as any unusualstrenuous exercise 24 hours prior to each experimental session. A singleblinded randomized, cross-over study was conducted. Participants wereasked to complete a total of three experimental sessions, separated by awashout period of 10 days, and to consume one of the three differenttypes of bread in random order. On each session, overnight fastedsubjects were asked to ingest 90 g (containing 50 g of carbohydrate) ofbread within 10-15 minutes (min) and subsequently drink 200 ml of water.Blood samples were collected at baseline (before bread ingestion) and at15, 30, 45, 60, 90 and 120 min post bread ingestion. Blood samples werethen centrifuged for 15 min at 4° C. at 1000 RPM and serum was stored inaliquots at −80° C. till analysis. Serum glucose, triglycerides (TG),total P, K and Mg were measured using the Vitros analyser 350 byOrtho-Clinical Diagnostics, Johnson & Johnson, N.Y.

Statistical Analysis

Experiment 1: Data related to triangle tests were analyzed by checkingthe minimum number of correct responses using a binomial table withp=0.05. Lawless H T & Heymann H (2010) Sensory Evaluation of Food,Springer. As for the acceptability test, a two-way analysis of varianceusing the GLM procedure of SAS® (version 9.02) was performed asdescribed in Lteif et al. (2009). In the statistical model foracceptability, the response variable was the specific acceptabilityvariable. Factors in the model were panelist and treatment (white,restored and fortified). Panelist was included as a random effect andtreatment as a fixed effect. Means were separated by Tukey's honestlysignificant difference test. For all data, significance was establishedat p<0.05.

Experiment 2: GI, which is the incremental area under the 2-hour bloodglucose response curve following a test food, compared to an equivalentcarbohydrate amount of a control food (white bread) consumed by the samesubject was calculated, as described in Wolever, T., Jenkins, D.,Jenkins, A., et al. (1991) The glycemic index: methodology and clinicalimplications. Am J Clin Nutr 54 (5), 846-854. Area under the curve (AUC)and the GI rating (%) of the test foods WP-R and WP-F were calculated,as in Brouns, F., Bjorck, I., Frayn, K., et al. (2005) Glycaemic indexmethodology. Nutr Res Rev 18 (1), 145-171. Paired t-tests were used tocompare differences between test foods (WP-R), (WP-F) and the controlfood (WP), at each time point. One-way ANOVA via Fisher's method wasthen used to detect statistical significance within the same bread type,at different time intervals. Repeated measures analysis of variance wasused to determine statistical significance with effects for bread type,time, and bread type by time interaction.

Results

Pita bread analysis (Table 5) showed that WP-R bread's P, K and Mgcontents were 84%, 60% and 200% higher than that of the WP,respectively. In the WP-F bread, P, K and Mg content were 260%, 230% and410% higher than that of the WP bread, respectively. P, K and Mgcontents of WP-F bread were almost double that of the WP-R bread.

TABLE 5 Phosphorus, potassium and magnesium content of the differentpita bread type Phosphorus (g/kg) Potassium (g/kg) Magnesium (g/kg) MeanSD Mean SD Mean SD WP 3.20 0.01 3.7 0.01 0.53 0.01 WP-R 5.90 0.00 5.90.23 1.60 0.04 WP-F 11.60 0.00 12.20 0.01 2.70 0.26 Results areexpressed as the mean and standard deviation (SD).

Experiment 1

Difference Test and Hedonic Acceptability

In the triangle difference test, thirteen correct answers out of the 24responses were needed to show a statistically significant difference.However, only 8 and 10 panelists responded correctly for the WP vs. WP-R(p>0.05) and WP vs. WP-F (p>0.05), respectively. Therefore, the triangletests did not detect any significant differences between the differenttypes of breads.

Moreover, the consumer acceptability test (Table 6) found no significantdifferences for most acceptability attributes (Overall Acceptability,Appearance, Colour, Odour and Flavour; p>0.05), except for texture(p<0.05) where the texture of WP was significantly more liked than theWP-F bread while no significant difference was detected between WP andWP-R breads or WP-R and WP-F breads.

TABLE 6 Hedonic acceptability variables for the different pita breadtype Acceptability Variables Overall Acceptability Appearance ColourOdour Flavour Texture Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD WP6.27 1.33 6.22 1.17 6.32 1.08 6.08 1.34 6.38 1.54 6.35^(a) 1.72 WP-R6.25 1.49 6.32 1.56 6.52 1.19 6.23 1.28 5.87 1.78 5.95^(ab) 1.84 WP-F6.07 1.33 6.28 1.21 6.40 1.39 6.12 1.53 5.85 1.62 5.42^(b) 1.71Significance 0.6010 0.8609 0.5422 0.7945 0.0657 0.0042 Results areexpressed as mean and standard deviation (SD). ^(a,ab,b)Mean with unlikesuperscripts are different (p < 0.05) as analyzed by paired t-test.

Experiment 2

Subjects Characteristics

Subjects' baseline characteristics are shown in Table 7. Subjects had amean age of 24.5 years and were mainly normal and overweight subjects(mean BMI 26 kg/m²). Baseline fasting levels of glucose, insulin, TG andtotal P indicate-that subjects were all within normal ranges.

TABLE 7 Baseline characteristics of the 12 subjects Mean SEM Age (yrs)24.50 1.02 Weight (kg) 83.92 3.35 Height (m) 1.79 0.01 BMI (kg/m²) 26.030.67 Fasting serum glucose (mg/dl) 94.00 3.28 Fasting serum insulin(μIU/mL) 6.679 1.92 Fasting serum triglycerides (mg/dl) 91.47 3.78Fasting serum phosphate (mg/dl) 3.79 0.19 Results are expressed as themean and standard error of the mean (SEM).

Postprandial Mineral Responses

Ingestion of the different types of pita bread was found to alterpostprandial level of the macro-minerals. Serum total P decreasedfollowing ingestion of all bread treatments and did not return tobaseline level by the end of the experimental session, except for thatof the WP-F. There was a significant difference between WP and WP-Ftreatments at 120 min. Additionally, changes in serum total P showedsignificant differences in serum total P between bread types (p<0.001)and across time intervals (p<0.001) (FIG. 8a ).

Postprandial serum Mg levels experienced a gradual increase followingthe ingestion of the different pita bread treatments. The increase seemsto have been halted at 30 min in the WP-R and at 45 min in the WP-Ftreatments (FIG. 8b ). There was a significant difference between WP andWP-F at 45 min. The changes in postprandial Mg were significant forbread types (p=0.031) and across time intervals (p=0.031). The magnitudeof postprandial Mg increase seems to be inversely related to the Mgcontent of the bread.

In contrast to that of Mg, changes in postprandial K levels experienceda decrease following the ingestion of the different bread treatments(FIG. 8c ). Again, these changes were found to be significantlydifferent for bread types (p=0.002) and across time intervals (p=0.001),whereby the magnitude of reduction seems to be more pronounced in theWP-R and WP-F treatments.

Postprandial Triglycerides and Glucose Responses

The pattern of changes in postprandial serum TG (FIG. 9a ) was found todiffer following the ingestion of the different types of breads. Changesin serum TG were significantly different for bread type (p<0.001) andtime (p=0.034) (FIG. 9a ), whereby WP-R and WF-R had lower levels of TGat several time points, though paired t-tests failed to detect anystatistical significance for these time points (p>0.05).

Postprandial serum glucose change (FIG. 9b ) was found to vary betweenthe different types of ingested breads. Analysis of variance revealedsignificant differences between bread types (p<0.030), time (p<0.001),and bread by time interaction (p<0.001). In brief, glucose levels peakedat 30-45 min post-ingestion. Thereafter, the rate of drop was faster inthe supplemented bread types (WP-R and WP-F) compared to the control(WP). Paired t-tests showed significant differences between WP and WP-Rat 30, 90, and 120 min; and between WP and WP-F at 15, 60, 90, and 120min. At 60 min onwards, WP-R and WP-F significantly retained the lowestmean serum glucose change compared to WP. In line with the changes inpostprandial glucose, the GI of WP-R and WP-F breads was 33% and 37%lower than that of the control bread (WP) (FIG. 10). The GI of WP-F(62.9±8.0; p<0.001) was significantly lower than that of control bread(WP), while that of the WP-R (67.3±12.5; p=0.208) failed to reachsignificance.

DISCUSSION

The palatability of pita bread was not affected by the addition ofmacro-minerals as indicated by the lack of differences in the triangleand acceptability tests, although a small difference in texture wasdetected between WP and WP-F breads in the latter test. However, thisdifference was not detected by the panelists in the triangle tests,where subjects usually tend to be more attentive to differences and moreanalytical in their response. This possibly indicates that when thebread is assessed in its “entirety” the differences are not major. Thisis in line with others, where the addition of potassium, calcium andmagnesium salts as replacements of NaCl did not yield any differences inappearance, texture and taste of brown bread. Thus, it can be concludedthat the addition of macro-minerals to white wheat flour in an amountcomparable to that found in whole wheat flour and even in quantitiesdouble that amount did not have any significant effect on theacceptability of pita bread.

The reduction in serum P following the ingestion of the different typesof bread was in line with others. This drop is believed to be mediatedby insulin that is known to stimulate glucose uptake into cells alongwith an intracellular shift of P to initiate glucose phosphorylation.The return of serum P to its baseline level at time 120 min in the WP-Ftype may have been related to its increased content of P while thefailure of serum P in the WP-R to return to baseline level may implythat its content of P was not sufficient to fully meet the needs ofintracellular phosphorylation.

On the other hand, the low postprandial levels of serum Mg insupplemented breads (WP-R and WP-F) as compared to that of WP may havebeen attributed to an enhanced clearance rate, especially since Mgclearance was reported to be dependent on insulin sensitivity that isknown to be enhanced by P ingestion. Additionally, improved insulinsensitivity may have also contributed to the postprandial changes inserum K between supplemented (WP-R and WP-F) and WP breads. Furthermore,glucose clearance and insulin sensitivity were reported to besynergistically related to Mg and K statuses. Accordingly, the increasedcontent of Mg and K in bread (WP-R and WP-F) may have further improvedtheir own clearance rate.

In the macro-mineral supplemented breads, the reduction in postprandialglucose starting at 60 min may have been brought by the capacity of theadded minerals to improve insulin sensitivity and thus glucoseclearance. This suggests that glucose clearance and insulin sensitivityat 60 min after meal ingestion depend on exogenous factors including P,Mg and K.

Likewise, the ability of added minerals to improve insulin sensitivitymay have been behind the observed reduction in serum TG of supplementedbreads (WP-R and WP-F). Phosphorus status was reported to be correlatedto a favourable lipid profile including increased HDL and decreasedserum TG levels. Moreover, Mg supplementation was also shown to improvepostprandial lipidaemic response in healthy subjects.

GI of supplemented breads relative to that of WP bread was reduced by 33to 37% for WP-R and WP-F, respectively. The magnitude of reduction in GIwas much higher than that observed in the micronutrient enriched steamedbread (about 25%), where glucose was used as a control. In summary,supplementation of white wheat flour with macro-minerals (P, Mg and K)did not affect the palatability of pita bread, while the GI was reduced.Consumption of about 500 g of WP-R, an excessive and high level outlierby any measure, would still be short of the upper limit for both P andMg.

Worldwide, wheat and wheat products per capita daily consumption isabout 180 g and this contributes to about 20% of total energy intakemainly in the form of bread and pasta. In Lebanon, bread intake, mainlyin the form of white wheat pita bread, was reported to be about 146 g/d.The high palatability of white bread, a high GI food, made it verypopular and a major contributor to the overall glycaemic load. Wholegrain cereals that have low GI are known to have a protective roleagainst the development of diabetes, abnormal lipid profile and obesity.However, the beneficial effect of whole wheat cereal products andspecifically wheat bran was not identified in terms of the exactcomponent that is behind these effects. This data indicates that thereduced GI of whole grain may be largely attributed to its content ofmacro-minerals, specifically potassium, K and Mg. It is worth notingthat the ingestion of 200 g of WP-R (1200 mg of P) or WP-F (2320 mg ofP) would still be below the upper limit of intake for P (4 g/d).

This example has successfully identified the potential beneficial roleof minerals in improving the glycaemic response of white bread andoverall glucose control in healthy male subjects. Even though thebeneficial effects of whole grains have been widely publicized, theadoption of diets rich in whole grains still faces resistance. Thismight be due, at least in part, to the low palatability of unrefinedcereal products. In this example, sensory results did not indicate anymajor effect of the experimental treatments on the quality of the bread,which is an encouraging outcome and indicative of the tolerance of breadquality to the addition of K and Mg. Results from the this example arepromising and may beget future research that investigates the long termeffect of mineral-supplemented bread on glycaemic status, and this maybe used as a tool for the prevention or management of diabetes and othercomponents of the metabolic syndrome

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

While the invention has been described in connection with variousembodiments, it will be understood that the invention is capable offurther modifications. This application is intended to cover anyvariations, uses or adaptations of the invention following, in general,the principles of the invention, and including such departures from thepresent disclosure as, within the known and customary practice withinthe art to which the invention pertains.

What is claimed is:
 1. A formulation of food products supplemented withmacro-minerals comprising: a food product including a carbohydrate thatlowers the glycemic response including Phosphorus (P) at an activeconcentration.
 2. The food product of claim 1, wherein the food productreduces the glycemic index by about
 30. 3. The food product of claim 1,wherein the food product reduces subsequent energy intake by about 30%.4. The food product of claim 1, further comprising Potassium (K) andMagnesium (Mg) at an active concentration.
 5. The food product of claim1, wherein Phosphorus is selected from the group consisting of:phosphorus-based acids, Monopotassium phosphate, phosphates, thephosphate ions, phospholipids, soluble salts of potassium,bisphosphonate, a hydroxybisphosphonate, a phosphonate, a phosphate, anaminomethylenephosphonic acid, and acidic peptide.
 6. The food productof claim 5, wherein the food product is a bread product and each Kg ofwhite flour contains between about 2.0 and 5.0 g MgCO₃ and between about5.0 and about 10.0 g of KH₂PO₄.
 7. A method of preventing weight gainand reducing waist circumference, comprising: delivering at leastbetween about 300 mg to about 500 mg of phosphorus with each meal,wherein each meal include about 300 to about 500 Kcal of carbohydrateover a period of time and increasing energy expenditure.
 8. The methodof claim 7, further comprising reducing the glycemic index by about 30.9. The method of claim 8, further comprising reducing the subsequentenergy intake by about 30%.
 10. The method of claim 9, furthercomprising delivering Potassium (K) and Magnesium (Mg) at an activeconcentration.
 11. A method of enriching a bread product comprising:restoring the levels of phosphorus (P), potassium (K), and magnesium(Mg) prior to processing and milling, whereby each Kg of white flourcontained between about 2.0 and 5.0 g of Mg and between about 5.0 andabout 10.0 g of K and P, and fortifying the bread product to double thepremilling levels of phosphorus (P), potassium (K), and magnesium (Mg).12. The method of claim 11, where the fortifying step comprises each Kgof white flour containing between about 5.0 and about 10 g of Mg andbetween about 20 and about 30 g of K and P.
 13. The method of claim 12,wherein the phosphorus percentage is increased by at least about 50% toabout 500%, the potassium percentage is increased by at least 50% toabout 500%, and the magnesium percentage is increased by at least 50% toabout 500%.